A new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered: a clinico-radiological and histomolecular characterisation

Diffuse midline gliomas (DMG) H3 K27-altered are incurable grade 4 gliomas and represent a major challenge in neuro-oncology. This tumour type is now classified in four subtypes by the 2021 edition of the WHO Classification of the Central Nervous System (CNS) tumours. However, the H3.3-K27M subgroup still appears clinically and molecularly heterogeneous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tended to have a better prognosis. To better study the role of these co-driver alterations, we assembled a large paediatric and adult cohort of 29 tumours H3K27-altered with co-occurring activating mutation in BRAF or FGFR1 as well as 31 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylation analysis. Interestingly, unsupervised t-distributed Stochastic Neighbour Embedding (tSNE) analysis of DNA methylation profiles regrouped BRAFV600E and all but one FGFR1MUT DMG in a unique methylation cluster, distinct from the other DMG subgroups and also from ganglioglioma (GG) or high-grade astrocytoma with piloid features (HGAP). This new DMG subtype harbours atypical radiological and histopathological profiles with calcification and/or a solid tumour component both for BRAFV600E and FGFR1MUT cases. The analyses of a H3.3-K27M BRAFV600E tumour at diagnosis and corresponding in vitro cellular model showed that mutation in H3-3A was the first event in the oncogenesis. Contrary to other DMG, these tumours occur more frequently in the thalamus (70% for BRAFV600E and 58% for FGFR1MUT) and patients have a longer overall survival with a median above three years. In conclusion, DMG, H3 K27 and BRAF/FGFR1 co-altered represent a new subtype of DMG with distinct genotype/phenotype characteristics, which deserve further attention with respect to trial interpretation and patient management. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-023-02651-4.


Introduction
Thanks to significant advances in genomics, the 2016 then-2021 World Health Organization (WHO) Classification of Tumours of the Central Nervous System (CNS) has defined tumour entities based on histological but also molecular features, like the driver genetic event [15,16].Diffuse midline gliomas (DMG) H3 K27-altered have now been identified as a new type of malignant gliomas which occur in the paediatric and adult populations, although with disparities according to the preferential location, i.e. brainstem in children and thalamus in adults [18,45].
DMG H3 K27-altered are either characterised by the substitution in histone H3 of the lysine at position 27 by a methionine (H3K27M), or the overexpression of EZHIP [3,42].Both mechanisms lead to Polycomb Repressor Complex 2 (PRC2) inhibition with a global loss of H3K27me3 [5,13], and consequently a major epigenetic and transcriptomic remodelling [1,3].According to different molecular and clinical parameters, including specific DNA methylation profiles, DMG H3 K27-altered appeared more heterogeneous than initially thought, and were further classified Extended author information available on the last page of the article in four subtypes: H3-3A K27-mutant (H3.3-K27M),H3C2 K27-mutant (H3.1-K27M),H3-wild-type (with EZHIP overexpression) or EGFR-altered [3,4,16,19,33].Despite this subdivision, we still observed some clinical and molecular heterogeneity within the most common H3.3-K27M subtype.We previously demonstrated that TP53 co-driver mutations are associated with a worse tumour response to radiotherapy and a poorer outcome [41] suggesting that additional molecular alterations can deeply modify the phenotype induced by the driver histone H3 mutation.Recently, we and others have described single cases or small studies of tumours with concomitant alterations of H3-K27M and mitogen-activated protein kinase (MAPK) pathway showing a possible longer survival compared to patients with DMG H3 K27-altered, BRAF and FGFR1 wild-type.In these studies, diagnoses ranged from H3.3-K27M BRAF V600E /FGFR1 MUT pilocytic astrocytoma or midline gangliogliomas grade 1-3 [11, 21-23, 26, 29, 44] to diffuse gliomas grade 4 [25,31].As BRAF and FGFR1 mutations are typical hallmarks of low-grade gliomas/glioneuronal tumours such as ganglioglioma or pilocytic astrocytoma, the co-occurrence of H3-K27 and these MAPK alterations makes diagnosis and grading difficult [30].In order to understand how these alterations could mitigate the prognosis of these neoplasms, we analysed (radiologic, histologic, genomic, transcriptomic and DNA methylation analyses) a larger DMG H3 K27-altered cohort, comprising 29 tumours harbouring BRAF V600E or FGFR1 MUT complemented by paediatric and adult cases from the literature.

Patients and tumour samples
The first part of the cohort is composed of 60 patients diagnosed with H3-K27M or EZHIP-overexpressing tumours harbouring a coding mutation in BRAF or FGFR1 genes, from the Necker Enfants-Malades/GHU-Sainte Anne Hospital/ Gustave Roussy center and Biomede 1 Trial (NCT02233049) (n = 29), or from other published cohorts (n = 31) [3,18,23,25,30,31,37].The control cohort includes patients with wild-type FGFR1/BRAF, in the above-mentioned cohorts of H3K27M or EZHIP-overexpressing DMG (flowchart in Supplementary Fig. 1, online resource).Tumour tissue and clinical data were collected under informed consent obtained from the parents or guardian according to the IRB approved protocol (CNIL 1176643).

Radiology analysis
All available radiology outcomes at diagnosis (CT and MRI) of patients with H3.3-K27M tumours from our cohort were reviewed centrally by three experts (VDR, NB, and JG).
Parameters specifically recorded were: radiological presentation (diffuse, circumscribed or nodular and diffuse), presence of contrast enhancement (yes/no) and calcifications (yes/no).For circumscribed tumours, the pattern at evolution was also analysed.

Histopathological analyses and immunohistochemistry
Formalin-fixed paraffin-embedded (FFPE) tissue samples for each patient were retrieved and Haematoxylin-Phloxine-Saffron (HPS)-stained slides were analysed by two experienced neuropathologists (PV and ATE) to confirm morphological diagnoses.Micro-calcifications were noted as present or absent as well as granular bodies, ganglion neurons, necrosis, and microvascular proliferation.Mitotic activity (per 2 mm 2 ) and tumour growth architecture were analysed within the inherent limits of a stereotaxic biopsy exploration.The latter was labelled as diffuse, compact tumoral areas or both.Morphological aspects were evaluated as ganglioglioma-like (GG-like), HGG with piloid astrocytic component or DMG-like.The infiltration pattern was also assessed by NF70 immunostaining (i.e.residual NF70 network or not).Immunostaining was performed from 3 μm-thick representative FFPE sections using a Dako OMNIS automate.The following primary antibodies were used: H3K27me3

DNA/RNA extraction and sequencing
DNA and RNA were extracted from frozen tumours using Allprep DNA/RNA kit (Qiagen) and were quantified using, respectively, the Qubit Broad Range double-stranded DNA assay (Life Technologies) or the Qubit RNA high sensibility (Life Technologies).When no frozen material was available for DNA methylation profiling, tumour DNA was extracted from formol-fixed paraffin-embedded (FFPE) block sections using dedicated protocols at Diagenode or Integragen.Targeted DNA sequencing (≥ 6000× coverage) or whole exome sequencing (≥ 130× coverage) was performed as previously described [10,41].WES was aligned with BWA according to GATK best practice guidelines, then Mutect2 was used for the DNA calling.RNAseq on DMG primary tumours was performed at Integragen (Evry, France).PolyA mRNA molecules were initially purified from at least 100 ng total RNA (NEBNext® Poly(A) mRNA Magnetic Isolation Module, NEB) and libraries were then prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB).Pairedend reads of 100 bp were generated on an Illumina NovaSeq reaching an average sequencing depth of 60 million reads.

DNA Methylation array processing
Genome-wide DNA methylation analysis was performed using either the Illumina HumanMethylation450 BeadChip (450 k) or EPIC arrays as previously published [2,3].Data were obtained from different platforms (DKFZ Heidelberg; Integragen; Diagenode; published data) and were analysed with R (v4.0.4).For t-Distributed Stochastic Neighbour Embedding (t-SNE) analysis, the minfi package was used to load idat files and preprocessed with the function preprocess.illumina for dye bias and background correction.Probes located on sex chromosomes or not uniquely mapped to the human reference genome were removed.Probes containing single-nucleotide polymorphisms or that were not present in both EPIC and 450 k methylation array were also eliminated.A batch effect correction was done with removebatchEffect function from limma package, to remove difference between formalin-fixed paraffin-embedded and frozen samples.The probes were sorted by standard deviation.The 10,000 most variable probes were used for subsequent clustering analysis and to compute the 1-variance weighted Pearson correlation between samples.The distance matrix was used as input in t-SNE from Rtsne package.For the second analysis, DNA methylation-based classification of CNS tumours from DKFZ-Heidelberg was used in order to predict the CNS tumour class based on the V12.7 of the classifier (www.molec ularn europ athol ogy.org).

Gene expression analysis
Reads were pre-processed using the nf-core RNAseq pipeline (v3.0), mapped to the reference genome GRC38/hg38 with the STAR tool (v2.6.1d),annotated with GENCODE v36 and counting was performed with the Salmon quantification tool (v1.4.0).Differential gene expression analysis was performed with the DESeq2 package (v1.30.0, minRep-licatesForReplace = 7, betaPrior = TRUE) with a threshold of 0.01 for Benjamini-Hochberg adjusted p value (adj-p).For gene set enrichment analysis (GSEA), hypergeometric tests were used to identify overrepresented gene sets from the MSigDB v7.4 database, amongst genes ranked by significance and fold-change in differential expression analysis, with Benjamini-Hochberg multiple testing correction using the package Clusterprofiler.Catalogues considered included Hallmark and C2.Differences were considered as significant when false discovery rate adj-q value was < 0.02.Gene expression comparison was evaluated with Wald test using DESeq2.

Univariate and multivariate survival analyses
Overall survival (OS) was estimated with the Kaplan-Meier method and median overall survival was computed using a log-rank test.OS was obtained from the post-diagnosis until death patient or last known information.The analysis was realised in Prism9 software.Multivariable Cox proportional hazards regression model on OS was performed including histone H3, BRAF, FGFR1, TP53 status, age at diagnosis and tumour location with R software using the function coxph() of the survival package (Version 3.2-13).

Statistical analyses
Distribution of age at diagnosis according to different parameters was accessed by Mann-Whitney test.Presence of macro-calcification, contrast enhancement, radiologic profile and sex ratio were evaluated by Fisher's exact test.Chi-square test for trend was used to evaluate tumour type and location.All statistic tests were performed using Prism 9 software (GraphPad).
We further analysed the genomic landscape of tumour for which material or data were available and observed that BRAF MUT or FGFR1 MUT was mostly associated with H3.3-K27M mutation but not H3.1-K27M mutation.One tumour harboured a FGFR1 MUT in the context of a DMG H3-K27 wild-type with EZHIP overexpression presenting an ACVR1 mutation (case #60).Second, BRAF and FGFR1 mutations were mutually exclusive, as only one tumour presented both hits (case #23; Fig. 1).However, no clonality information was available for this case to confirm subclonality of the two mutations.Finally, we observed that in 90% (9/10) of FGFR1 MUT cases for which we had the information, FGFR1 mutation was clonal to H3-3A mutation with a similar variant allele frequency suggesting a role in the early steps of oncogenesis of these tumours.It was less frequently the case for patients with DMG H3-K27M BRAF V600E with only 33% (2/6) tumours where BRAF mutation appeared clonal to H3-K27M mutation.In addition, H3-K27 FGFR1 MUT tumours presented often other hits in the MAPK pathway with NF1 (13/31; 42%) or PTPN11 in (3/22; 13.6%) as the topmost mutated genes (Fig. 1).Additional MAPK-activating mutations seemed to be less frequent for BRAF MUT tumours with 1/12 (8.3%) case harbouring an NF1 mutation.TP53 mutations were found in 5% (1/20) and 9.3% (3/32) of the BRAF MUT and FGFR1 MUT tumours, respectively but not PPM1D (Fig. 1).For one H3.3-K27MFGFR1 MUT patient, TP53 mutation was clonal to H3.3-K27M and it was subclonal for the other case for which information was available.In addition, ATRX was mutated, or its expression was lost on IHC in 59.3% (16/27) of FGFR1 MUT DMG, albeit never in BRAF MUT tumours, and this was not associated to TP53 MUT in all but one case (#24).A mutation in a member of the PI3K/AKT/mTOR signalling pathway was present in 19% (4/21) of H3-K27M-FGFR1 MUT tumours (Fig. 1).

DNA methylation profiling distinguishes a subgroup of DMG H3 K27-altered with MAPK-activating mutations
Given the disparities between DMG_K27-BRAF/FGFR1 and classical DMG H3K27-altered, we hypothesised that DMG H3 K27-altered with MAPK-activating mutations might correspond to either (i) a new subtype of DMG or (ii) atypical aggressive MAPK-driven low-grade gliomas/ glioneuronal tumours.To test these hypotheses, we analysed the DNA methylation profile of the whole cohort.Based on the Heidelberg DNA methylation brain classifier V12.8, 54% (7/13) of BRAF MUT and 67% (10/15) of FGFR MUT tumours classified as DMG_K27 and the remaining corresponded to other classes or were undefined (score < 0.9) (Supplementary Fig. 3a, online resource), which was consistent with the hypothesis that DMG H3 K27-altered with MAPK-activating mutation constitute a unique subtype of DMG.In contrast, all DMG H3.3-K27M BRAF WT /FGFR WT clustered as DMG_K27.We then performed an unsupervised clustering based on DNA methylation profiles of these samples together with reference gliomas from the literature [2].All

BRAF and FGFR1 mutational status are prognostic in paediatric and adult DMG H3 K27-altered
TP53 mutations and BRAF V600E /FGFR1 mutations are mostly mutually exclusive in DMG.In order to avoid the confounding effect of TP53 mutations on the outcome of H3.3-K27M DMG without MAPK-activating alterations, we stratified patients on Histone H3 and TP53 genotypes of both subgroups in Kaplan-Meier overall survival (OS) analyses.This analysis showed a significant better OS for paediatric and adult DMG H3-K27 patients with activating BRAF (median OS 37 mo.) or FGFR1 mutations (median OS 36 mo.) compared to DMG H3.3-K27M TP53 WT (median OS 12 mo.) and other DMG subtypes (Fig. 5a; p value < 0.0001, global log-rank test).Further, we showed that there was no impact of histopathological features such as microvascular proliferation, necrosis or mitotic index, on the OS of patients with BRAF or FGFR1-mutated DMG (supplemental Fig. 2f-h, online resource).To pursue, we performed a multivariable analysis to evaluate the association of Histone H3, BRAF, FGFR1 and TP53 mutational status, age at diagnosis and tumoral location with survival.As expected from previous publications, histone H3 and TP53 status were significantly associated with OS (Table 1) [4,41].Age was significantly associated with prognosis, but its overall impact was only marginal compared to other variables.We also showed that BRAF V600E (HR: 0.2132, 95% CI 0.1098-0.4140,p value = 5.03e-06) and FGFR1 MUT (0.3414, 95%CI 0.1963-0.5939,p value = 0.0001) are strong and independent prognostic markers in H3K27-altered DMG (Table 1).

Clinical disparities in paediatric and adult patients with DMG H3-K27M BRAF/FGFR1-mutated
We next compared other clinical parameters.The sex ratio was balanced in DMG with MAPK-activating mutations (Supplementary Fig. 4a, online resource).However, we identified a significant difference in age at diagnosis between H3.3-K27M DMG, BRAF MUT and FGFR1 MUT DMG (Fig. 5b).More precisely, H3.3-K27M BRAF V600E DMG developed only in children (< 18 years) with a median age at onset of 7.2 years lower than DMG H3.3K27M with 9.85 years (Fig. 5b; p value = 0.0123, Mann-Whitney test) but similar to DMG H3K27-altered paediatric cases with 7.6 years in a restricted paediatric cohort (supplementary Fig. 4b, online resource).In contrast, patients with FGFR1 MUT have a significant higher age at diagnosis compared to those FGFR1 WT with median of 14.8 and 9.8 years respectively (Fig. 5b; p value = 0.0001, Mann-Whitney test).The age at diagnosis of H3-K27M DMG can vary according to initial tumour locations, with a higher age of onset for thalamic versus pontine tumours [18,34].We found this difference of age according to tumour location and the false discovery rate (FDR q) are indicated in each plot in DMG H3.3-K27M and in DMG BRAF V600E but not in DMG FGFR1 MUT (Supplementary Fig. 4c, online resource).Finally, DMG with BRAF and FGFR1 mutations were significantly more frequent in the thalamus compared to DMG H3.3-K27M (Fig. 5c; 70% (16/23) and 58% (21/36), respectively versus 28% (55/199); p value = 0.0004, and p value < 0.0001, Chi-square test for trend).No variation in term of OS, age at diagnosis and tumour location was noted according to FGFR1-mutated variant: FGFR1 N546K/D versus FGFR1 N656E (Supplementary Fig. 5, online resource).

H3.3-K27M mutation occurs prior to BRAF V600E during oncogenesis
We next wondered if the sequence of appearance of mutations was identical in these tumours, and more largely if they correspond to (i) DMG (H3-K27M as a first hit) or (ii) atypical aggressive low-grade gliomas (BRAF V600E as first hit).To address this question, we analysed BRAF copy number variation (CNV) by digital droplet Polymerase Chain Reaction (ddPCR) on genomic DNA from one BRAF V600E H3.3-K27M tumour (case #2) and from a H3.3-K27M BRAF WT clone derived from this primary tumour in vitro (Supplementary Fig. 6, online resource).The BRAF WT clone had two BRAF alleles and thus resulted from the re-amplification of an ancestral H3.3-K27M-only clone, but not from a genetic loss of the BRAF V600E allele, demonstrating that, in one DMG H3.3-K27M BRAF V600E , H3.3-K27M was the first hit.Interestingly, DNA methylation profiles from this in vitro amplified BRAF WT H3.3-K27M ancestral clone from case #2 and also from a tumour relapse enriched in H3.3-K27M BRAF WT clone from patient #7 (initially diagnosed with DMG H3.3-K27M BRAF V600E ), clustered with DMG H3-K27 with MAPK alterations instead of classical DMG H3-K27 (Supplementary Fig. 3b, online resource).

DMG H3.3K27M with MAPK alterations show a transcriptomic signature of senescence with up-regulation of CDKN1A (P21)
In order to investigate the possible specificities of the DMG H3.3-K27M with BRAF MUT or FGFR1 MUT , we compared their transcriptome to regular DMG H3.3-K27M TP53 WT separately.We found 676 significantly up-regulated and 633 down-regulated genes in the contrast H3.3K27M BRAF WT versus H3.3-K27M BRAF V600E DMG and 228 up-regulated and 274 down-regulated genes in the contrast H3.3-K27M FGFR1 WT versus H3.3-K27M FGFR1 MUT DMG (adj.p value ≤ 0.001) (Supplementary Fig. 7a, b, online resource).Ninety-four up-regulated and 111 down-regulated genes were common to the two comparisons.Using gene set enrichment analyses (GSEA), we observed an enrichment for MAPK signalling and PI3K/AKT/MTOR signalling signatures in both BRAF MUT and FGFR1 MUT DMG (Fig. 5d; Supplementary Fig. 7e, f, online resource) as well as angiogenesis and hypoxia signatures (Supplementary Fig. 7 g-j, online resource).In addition, transcriptomic signatures highlighted activation of senescence and P53 signalling pathway in both comparisons (Fig. 5d).P53 protein is a tumour suppressor implicated in the permanent cell cycle arrest by inducing senescence or apoptosis in response to stress like oncogene activation [6,14].TP53 pathway activation was validated at the protein level by immunohistochemistry (IHC) in 71% (10/14) of DMG H3.3-K27M TP53 WT with BRAF/FGFR1 alterations, showing heterogeneous, weak to strong TP53 staining.Moreover CDKN1A, encoding the senescence marker P21, was overexpressed at the RNA level in both BRAF MUT and FGFR1 MUT gliomas compared to those only H3.3-K27M mutated (adj.p value < 0.0001) (Supplementary Fig. 7 k online resource).CDKN2A, which encodes the tumour suppressor P16, was overexpressed only in BRAF-mutant gliomas (Supplementary Fig. 7 l online resource; adj.p value = 0.0048).As a whole, based on transcriptome and IHC, DMG H3-K27 with BRAF MUT / FGFR1 MUT were characterised by a senescence programme, likely induced trough a P16/P21-P53 axis.

Discussion
The recent description of 'DMG, H3 K27-altered' and its sub-classification into four molecular subgroups does not capture completely the diversity of this disease [16].Our data support the individualization of an additional new subtype of DMG with distinct histological, radiological, clinical, genomic, transcriptomic and epigenetic features that we provisionally termed DMG, H3 K27 and BRAF/FGFR1 coaltered (DMG_K27-BRAF/FGFR1) which may represent 20% of DMG H3 K27-altered.Using unsupervised analysis of DNA methylation tumour profiles, DMG_K27-BRAF/ FGFR1 formed a specific cluster, separated from other DMG_K27 gliomas, others adult/paediatric diffuse gliomas, low-grade glial/glioneuronal tumours and more specifically BRAF V600E -mutated ganglioglioma even midline located.
This highlights a possible distinct cell of origin for DMG_K27-BRAF/FGFR1, able to exhibit a mixed glial and neuronal differentiation, mostly noticeable in the BRAF subclass [2,39].Schüller et al. did not mention any of these phenotypes in DMG H3-K27M FGFR1 MUT , due to the limited number of tumours [31].The analysis of the H3.3-K27M ancestral clone derived from a DMG BRAF V600E H3.3K27M harboured this same DNA methylation profile, confirming that the specific DNA methylation profile of DMG_K27-BRAF/FGFR1 is not a strict consequence of MAPK alterations.The fair discrimination of tumours on the tSNE based on the type of the secondary MAPK mutation (i.e.BRAF vs. FGFR1) even suggests that this entity could be further subdivided.Genotype-morphotype correlations support the distinction from classical DMG, H3 K27-altered and from glial/ glioneuronal tumours MAPK-altered: (i) whilst ependymal differentiation has been described in rare DMG H3-K27 [36], a mixed glioneuronal differentiation associated with CD34 positivity and eosinophilic granular bodies or a piloid differentiation are not yet described; (ii) only an exceptional subset of ganglioglioma grade 1 present FGFR1 alteration, more characteristic of other glioneuronal tumours [24] and (iii) the existence of true malignant transformation in ganglioglioma is a matter of debate.The majority of reported cases were published before the advent of molecular biology, reclassified in a wide spectrum of CNS WHO tumour types without a distinct methylation class [28] or more interestingly were midline-located with a co-occurring BRAF and H3-K27M mutations [11, 21-23, 26, 29, 30, 44].In the unified methylation class that we describe, the radiological and histopathological presentations are thus highly heterogeneous including tumours with mixed glioneuronal or pilocytic differentiation, and do not always fulfil a strict diagnostic criterion of DMG.Indeed, these tumours are less diffuse, with a frequent nodular to circumscribed radiological aspect (91% for BRAF MUT and 78% for FGFR1 MUT DMG) and calcifications.
Several other clinical and biological characteristics support the individualization of this new entity from classical DMG, H3 K27-altered.First, OS is significantly different from classical DMG_K27, with a median around three years for both FGFR1 MUT and BRAF MUT H3.3K27M DMG.
Moreover, our multivariate analysis demonstrates for the first time that the presence of these mutations is an independent prognostic factor for improved OS in DMG_K27.Previously, Picca et al. and Schüller et al. showed in a small populations (n = 6 or n = 7), by univariate analysis and without taking into account TP53 status, that DMG patients with FGFR1 MUT have a better survival [25,31].The identification of a new subtype of DMG H3 K27-altered with longer survival is also a step forward for clinical research, highlighting the need for patient's stratification in trials or at least molecular documentation of the cases.
Patients from Necker/Gustave Roussy cohort with BRAF/ FGFR1-mutated DMG_K27 received, over a large period of time, quite heterogeneous treatment which did not allow specific statistical conclusion.It thus remains to be defined whether these patients could respond to a targeted therapy against BRAF V600E or FGFR1.
Another meaningful difference is that DMG_K27-BRAF/ FGFR1 are more frequent in the thalamus than the brainstem compared to DMG from the H3.3-K27M subtype.The age at diagnosis also differs according to the presence of the MAPK alteration.The age at onset for FGFR1 MUT   DMG is significantly higher (median 14.8 years) and to our knowledge no adults were affected by a H3.3-K27M BRAFmutated gliomas.In this new subtype, some heterogeneity remains present at various level between DMG H3K27M BRAF or FGFR1-mutated.This finding cannot be presently explained, but it may also point towards different oncogenesis, which could be individualised in the future studies.
We also investigated gene expression in these tumours and observed a senescence signature including an up-regulation of CDKN1A (P21) specific to DMG_K27-MAPK which is usually more present in paediatric LGGs.LGGs are characterised by an over-activated MAPK signalling in consequence to oncogenic alteration of BRAF or FGFR1 [15] and the main hypothesis for a slow tumour evolution in LGG is based on the induction of oncogene-induced senescence which gives a growth advantage in a restricted window during brain development [7,8,27,35,40].Senescence triggered via the P53/P21 axis could in part explain the slower tumour evolution in DMG_K27-MAPK.Of note, the DMG_K27-BRAF/FGFR1 share other characteristics with LGGs like calcifications and preferential association of mutations: FGFR1 with NF1, PI3KCA, PTPN11 [9,17,32,38].We also demonstrated in one case from this new DMG subtype harbouring BRAF V600E , that H3.3K27M was the first mutational event in its oncogenesis.Thus, BRAF and FGFR1 mutations would be secondary driver events in the oncogenesis of these tumours and could give a proliferative advantage to the H3.3-K27M ancestral clone in a specific developmental window similarly to paediatric LGGs.Extending the analysis of sequential acquisition of mutations in DMG_K27-MAPK oncogenesis will be essential for designing future therapeutic interventions.
In conclusion, we have identified a fifth subtype of DMG, H3 K27-altered that we named 'DMG H3 K27 and BRAF/ FGFR1 co-altered' (DMG_K27-BRAF/FGFR1), which harbours specific clinical and biological characteristics.We hypothesise that the better OS of DMG_K27-BRAF/FGFR1 compared to other DMG_K27 could be the result of both a specific cell origin and the oncogene-induced senescence.Individualization of this subtype is of importance for the interpretation of trials and affected patients may deserve specific treatment strategies.

Fig. 1
Fig. 1 Clinical and molecular characteristics of the patient cohort of DMG H3-K27M with BRAF and FGFR1 mutations.Overview of the clinical and molecular annotations of 60 paediatric and adult DMG H3-K27 patients presenting BRAF or FGFR1 mutations.Cases are presented in columns and genes status in rows.Age is reported

Table 1
Multivariable Cox proportional hazards regression model for the OS of patients with H3-K27M DMG CI confidence interval, WT wild-type